Flow cytometry is a powerful tool used for analysis of particles and cells in a myriad of applications primarily in bioscience research and medicine. The analytical strength of the technique is in its ability to parade single particles (including bioparticles such as cells, bacteria and viruses) through the focused spot of light sources, typically a laser or lasers, in rapid succession, at rates up to tens of thousands of particles per second. The high photon flux at this focal spot produces scatter of light by a particle and or emission of light from the particle or labels attached to the particle that can be collected and analyzed. This gives the user a wealth of information about individual particles that can be quickly parleyed into statistical information about populations of particles or cells.
In traditional flow cytometry, particles are flowed through the focused interrogation point where a laser directs a laser beam to a focused point that includes the core diameter within the channel. The sample fluid containing particles is focused to a very small core diameter of around 5-50 microns by flowing sheath fluid around the sample stream at a very high volumetric rate on the order of 100-1000 times the volumetric rate of the sample. This results in very fast linear velocities for the focused particles on the order of meters per second. This in turn means that each particle spends a very limited time in the excitation spot, often only 1-10 microseconds.
In a conventional flow cytometer there are analytical tools and/or methods needed to track full system and subsystem performance. Subsystems that can fail in a flow cytometer can include optics, electronics, and fluidics either independently or collectively. Traditionally, flow cytometry data acquisition and/or diagnostics software comes with a mode for measuring the immediate system performance and comparing it with a previous day(s) performance. These performance tests often use a cocktail of beads with known fluorescent characteristics. The performance test will use these beads to make a series of measurements including coefficient of variation of a population of ‘bright’ fluorescent beads, optical background, and quantum efficiency of the detection channel. By monitoring these values and how they change, it can be determined when an instrument is no longer functioning within specification and should be serviced. The person servicing the instrument may run tests on the optics, electronics, and fluidics; the failure mode is then determined through process of elimination or isolation of variables.
Unfortunately, one of the biggest difficulties in servicing flow cytometers is that most measured parameters are derived from convoluted inputs of the optics, electronics, and fluidic systems. Techniques for isolation of many optical and electronic components exist. Due to the microfluidic nature of the fluidic system, very few sensors and tests are available to isolate and determine the health and/or accurately measure the flow profile of the fluid delivery system. For this reason, optics and electronics are tested and only if the problem isn't solved is the fluidic system tested. Beyond measuring steady-state pressure or investigating for leaks, testing of fluidics usually includes swapping in and out various components in the hopes of finding solutions. Flow cytometers with multiple laser beams are especially sensitive to pressure fluctuations within the fluid delivery system with fluctuations well below 1% of the total operating pressure causing coefficient of variation broadening in the optical data. In this situation a person would be called to fix the coefficient of variation broadening in the optical data and the testing begins at the optical and electronic interfaces.
As such, there is a need to be able to detect steady state and dynamic irregularities or failures in the fluidic systems for flow cytometers in isolation, to decouple fluidics from optical and electronic subsystems without having to run failed experiments and then troubleshoot various subsystems before the fluidics system can even be considered. Such a detection system can be used for both troubleshooting a broken fluidic systems as well as helping adjust a working fluidic system to meet the intended specifications.